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1 A Pharmacologic Activator of Endothelial KCa Channels Increases Systemic Conductance 2 and Reduces Arterial Pressure in an Anesthetized Pig Model 3 4 5 Ramesh Mishra1,4*, Jamie R. Mitchell5*, Carol Gibbons-Kroeker1,3,4,6, Heike Wulff7, Israel 6 Belenkie,2,3,4, John V. Tyberg1,2,4 and Andrew P. Braun1,4 7 8 Depts. of 1Physiology & Pharmacology, 2Cardiac Sciences and 3Medicine, Cumming School of 9 Medicine and 4The Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, 10 Alberta, Canada, 5Dept of Physiology, Faculty of Medicine and Dentistry, University of Alberta, 11 Edmonton, Alberta, Canada, 6Dept. of Biology, Ambrose University College, Calgary, Alberta, 12 Canada, 7Dept. of Pharmacology, University of California-Davis, Davis, CA, USA 13 14 * These authors contributed equally to this study 15 16 Running title: Hemodynamic effects of an endothelial KCa channel activator 17 18 Key words: Endothelium, pressure, conductance, , KCa channel 19 20 21 Correspondence should be addressed to: 22 Andrew Braun, Ph.D., 23 Dept of Physiology and Pharmacology, 24 Cumming School of Medicine, 25 University of Calgary 26 3330 Hospital Dr NW, Calgary, Alberta, Canada T2N 4N1 27 E-mail: [email protected] 28 Phone: (403) 220-8861 29 2

30 Abstract 31 SKA-31, an activator of endothelial KCa2.3 and KCa3.1 channels, reduces systemic blood 32 pressure in mice and dogs, however, its effects in larger mammals are not well known. We 33 therefore examined the hemodynamic effects of SKA-31, along with sodium nitroprusside 34 (SNP), in anesthetized, juvenile male domestic pigs. Experimentally, continuous measurements 35 of left ventricular (LV), aortic and inferior vena cava (IVC) pressures, along with flows in the 36 ascending , carotid , left anterior descending coronary artery and renal artery, were 37 performed during acute administration of SKA-31 (0.1, 0.3, 1.0, 3.0 and 5.0 mg/ml/kg) and a 38 single dose of SNP (5.0 µg/ml/kg). SKA-31 dose-dependently reduced mean

39 (mPAO), with the highest dose decreasing mPAO to a similar extent as SNP (-23±3 and -28±4 40 mmHg, respectively). IVC pressure did not change. Systemic conductance and conductance in 41 coronary and carotid increased in response to SKA-31 and SNP, but renal conductance 42 was unaffected. There was no change in either LV (SV) or rate (versus the 43 preceding control) for any infusion. With no change in SV, drug-evoked decreases in LV stroke 2 44 work (SW) were attributed to reductions in mPAO (SW vs. mPAO, r = 0.82, P < 0.001). In

45 summary, SKA-31 dose-dependently reduced mPAO by increasing systemic and arterial

46 conductances. Primary reductions in mPAO by SKA-31 largely account for associated decreases 47 in SW, implying that SKA-31 does not directly impair cardiac contractility. 48 49 50 Abbreviations: G, conductance; HR, ; IVC, inferior vena cava; KCa channel, calcium- + 51 activated K channel; ; mPAO, mean aortic pressure; mPIVC, mean inferior vena caval pressure;

52 PBS, phosphate-buffered saline; PLVED, left ventricular end-diastolic pressure; SKA-31, 53 naphthol[1,2-d]thiazol-2-ylamine; SNP, sodium nitroprusside; SV, stroke volume; SVR,

54 systemic ; SW, stroke work; VolD, volume of distribution 55 3

56 1. Introduction 57 The vascular endothelium plays a critical role in the regulation of and 58 blood flow distribution by controlling the intraluminal diameter of conduit and small resistance 59 arteries. This dynamic regulation occurs via the activation of distinct vasodilatory mechanisms in 60 the endothelium that reduce contractile tone in the surrounding vascular smooth muscle, leading 61 to increased intraluminal diameter, arterial conductance and blood flow. Major pathways 62 contributing to endothelium-dependent include the de novo synthesis of nitric oxide, 63 prostacyclin and the generation of a hyperpolarizing electrical signal that acts on vascular 64 smooth muscle. Endothelium-dependent hyperpolarization (EDH) is generated primarily via the 65 activation of endothelial small- and intermediate-conductance, Ca2+-activated K+ channels 66 (KCa2.3 and KCa3.1 channels, respectively) and is transmitted via myoendothelial gap junction 67 connections to the adjacent smooth muscle, where it causes membrane hyperpolarization and 68 reduced Ca2+ influx via voltage-gated Ca2+ channels. Small-molecule activators of KCa2.3 and 69 KCa3.1 channels evoke direct hyperpolarization of endothelial cells [1-5], relax myogenically 70 active resistance arteries [1,6] increase coronary flow in isolated heart preparations [7] and lower 71 blood pressure in normo- and hypertensive mice [2,5]. In conscious dogs, bolus administration of 72 a KCa channel activator transiently lowers systemic blood pressure [4]. In contrast, genetic 73 knockout of endothelial KCa channels in mice leads to elevated systemic blood pressure and 74 impairs or abolishes stimulus-evoked vasodilatory processes in isolated arteries and tissues [8]. 75 Endothelial KCa channel activity may also be important in disease settings, as a KCa channel 76 activator is able to restore agonist-evoked vasodilatory responses in the coronary circulation of a 77 rodent model of type II diabetes exhibiting endothelial dysfunction [9]. 78 To advance our knowledge of the in vivo cardiovascular effects of endothelial KCa 79 channel activators, the goal of the present study was to investigate the systemic hemodynamic 80 effects of SKA-31, a recently described, second-generation KCa channel activator [2], in a large 81 animal model, the anesthetized, instrumented pig. Our results demonstrate that bolus intravenous 82 injections of SKA-31 dose-dependently lower mean aortic pressure and increase systemic 83 conductance to levels comparable to those elicited by the sodium nitroprusside 84 (SNP). SKA-31 increased arterial conductance in coronary and carotid arteries, indicating that 85 SKA-31 may have broad vasodilatory action in the vasculature. Neither SKA-31 nor SNP 86 appeared to directly alter . In summary, our data demonstrate that SKA- 4

87 31 effectively lowers systemic blood pressure and increases arterial conductance in the 88 peripheral circulation of the anesthetized pig. These observations suggest that SKA-31 may also 89 be an effective vasodilator in the human vasculature. 90 91 2. Methods and Materials 92 The experimental protocols used in this study were approved by the University of 93 Calgary Animal Care Committee, and conform to the NIH-published Guide for the Care and Use 94 of Laboratory Animals (8th edition, 2011), and are further consistent with those of the American 95 Physiological Society. 96 97 2.1 Animal preparation 98 Seven male domestic pigs (25-30 kg body weight, average weight 27 kg, 16-18 weeks of 99 age) were studied. Pigs were pre-medicated with an intramuscular injection of ketamine 100 hydrochloride (600 mg), fentanyl citrate (2 mg), and midazolam (10 mg). A 20-gauge catheter 101 was inserted into an ear vein and anesthesia was induced with sodium thiopental (25 mg/kg). 102 Anesthesia (level 3) was maintained with a continuous intravenous (I.V.) infusion containing a 103 mixture of fentanyl citrate (0.04 mg/ml), midazolam (0.025 mg/ml) and ketamine hydrochloride 104 (0.3 mg/ml) at a rate of 100 ml/hour. Both isoflurane (less than 1% in the ventilator) and 105 lidocaine (3 bolus intravenous administrations, 1 mg/kg, 5 min apart, followed by an I.V. 106 infusion of 0.75 - 1.0 mg/min) were used as required. The drug infusion rates were adjusted as 107 necessary to ensure deep sedation without spontaneous respiratory effort. The animals were 108 intubated with a cuffed endotracheal tube and ventilated with constant-volume ventilator 109 (Harvard Apparatus, Millis, MA) with a 50% oxygen -50% nitrous oxide mixture. Tidal volume 110 and respiratory rate were adjusted to maintain physiological values of blood gases and pH in

111 accordance with recommended ventilation parameters for large animals [10]. PaCO2 was 112 maintained between 35 and 45 mmHg. 113 A median sternotomy was performed and the were delivered from the pericardium 114 through a base-to-apex incision. Sonomicrometry crystals (Sonometrics, London, ON) were 115 implanted in the left ventricular endocardium and mid-wall of the septum to measure the minor- 116 axis septum-to-left ventricular free wall and left ventricular antero-posterior dimensions [11-13]. 117 Ultrasonic flow probes (Transonic Systems, Ithaca, NY) were placed on the ascending aorta, 5

118 descending aorta (just above diaphragm), inferior vena cava (IVC) (just above the diaphragm), 119 right carotid artery, left renal artery, and left anterior descending coronary artery. Thin walled 7- 120 French fluid-filled catheters connected to pressure transducers (model P23 ID; Statham Gould,

121 Oxnard, CA) were inserted into the left (LV) (PLV; retrograde through the left carotid

122 artery), aorta (PAO; retrograde through the right femoral artery) and IVC (PIVC; through the right 123 jugular vein). An intravenous line was placed in the left external jugular vein for volume loading 124 (PentaspanTM, 10% pentastarch in 0.9% NaCl) to replenish fluid loss during surgery. A thin- 125 walled catheter was connected to the intravenous line for bolus infusions. Arterial samples for 126 blood-gas analysis were obtained from a side-port on the aortic catheter. Body temperature was 127 monitored with a rectal thermometer. After instrumentation, the heart was returned to the 128 pericardium, which was closed with individual sutures, taking care not to compromise pericardial 129 volume [14]. A single-lead electrocardiogram (ECG) was recorded. 130 131 2.2 Experimental protocol 132 Simultaneous pressure, dimension and flow measurements were recorded at baseline and

133 during each intervention. After stabilization at an LV end-diastolic pressure (PLVED) of ~10 134 mmHg (11 ± 1 mmHg), control data were collected for 60 s, immediately preceding a 5-min 135 recording period, during and after drug infusion. Each 20 ml infusion was delivered over a 60-s 136 period and proceeded in ascending order of SKA-31 dosage (0.1, 0.3, 1.0, 3.0, and 5.0 mg/ml/kg) 137 followed by a single dosage of sodium nitroprusside (SNP; 5.0 µg/ml/kg). Washout and recovery 138 periods of 15-20 min were interposed between drug infusions. At the end of the experiment, the 139 animals were sacrificed by a bolus KCl injection and the positions of the sonomicrometry 140 crystals within the myocardium were verified. 141 SKA-31 was synthesized and tested for identity and purity (NMR and HPLC/MS) as 142 previously described [2]. SKA-31 was dissolved in a vehicle solution comprised of Cremophor 143 EL (10% v/v) and phosphate-buffered saline (PBS) (90% v/v). Briefly, an aliquot of Cremophor 144 EL was first heated in a beaker on a magnetic stir plate to a temperature of ~600C. The desired 145 amount of solid SKA-31 was then added to the heated Cremophor EL liquid as it was being 146 stirred. Once the added SKA-31 had dissolved completely, heating was stopped and stirring was 147 maintained. The first few milliliters of PBS were then added slowly to the SKA-31/Cremophor 148 EL solution and the remaining amount was added more quickly. The final SKA-31 solution was 6

149 allowed to cool to room temperature with continuous stirring and appeared slightly yellowish. 150 Solutions of SKA-31 in Cremophor-EL/PBS were freshly prepared for each experiment. 151 152 2.3 Data analysis 153 The conditioned signals were passed through a low-pass filter (100 Hz) and were 154 digitized and recorded at 100 Hz (Sonometrics Corp. acquisition system, London, ON). The 155 digitized data were analyzed on a personal computer using custom software (CV Works, 156 Calgary, AB) developed in our laboratory. Baseline and control data are expressed as mean 157 values for the 60-s period immediately preceding each infusion event. All data associated with 158 administration of drug or control solutions were extracted at the time of greatest decrease in

159 mPAO. If mPAO did not change by at least 5 mmHg during a given intervention, acquired data 160 points were averaged for the first 60 s of that period.

161 Systemic conductance (Gsystemic, the reciprocal of systemic vascular resistance, SVR) was

162 calculated as mean aortic flow / (mPAO – mPIVC) and expressed as a percent change from the

163 preceding control value. Carotid conductance (Gcarotid), renal conductance (Grenal) and coronary

164 conductance (Gcoronary) were expressed similarly and calculated by respectively substituting mean 165 carotid, renal and coronary flow for mean aortic flow. LV stroke work (SW) was calculated as

166 LV stroke volume (SV) x [mean PLV (systolic) – PLVED], where mean PLV (systolic) was

167 calculated as PAO (diastolic) + 2⁄3 [PAO (systolic) – PAO (diastolic)]. As an index of LV end-

168 diastolic volume, LV area (ALVED), was calculated as the product of the 2 minor-axis LV

169 dimensions [15,16]. SW and ALVED values following drug infusions are expressed as the percent 170 change from the preceding control values determined using the same calculations. 171 172 2.4 Measurements of SKA-31 Concentration in Plasma 173 Blood samples (~2 ml) were taken via a catheter inserted into the left external jugular 174 vein at various intervals following drug infusion at the same site. Samples were collected in 175 heparinized tubes to prevent coagulation and centrifuged at 400 x g for 20 min at 4oC. The 176 resulting supernatants were then stored at -80oC prior to analysis. Plasma samples were then 177 processed and analyzed in duplicate by HPLC/MS as recently described [4] and SKA-31 178 concentrations were determined from a standard curve. A semi-logarithmic plot of SKA-31 179 plasma concentrations vs. time was fitted with the following equation: 7

180 SKA-31 Plasma Concent. = C0 * exp(-ket)

181 Where C0 = the maximal initial concentration of SKA-31 in the plasma calculated from the y-

182 intercept of the fitted line, ke is the rate constant and t is the time interval following SKA-31

183 infusion. The volume of SKA-31 distribution (VolD) was calculated as follows:

184 VolD = SKA-31 dosage/C0 185 186 2.5 Statistical analysis 187 Statistical comparisons were performed using SigmaPlot (Systat Software, Inc. 2012). In

188 Figure 8, a linear correlation was calculated for the percentage changes for mPAO and stroke

189 work during saline, vehicle, and all drug infusions (y = y0+a * x). The Student’s paired t-test was 190 used to test for the significance of changes between a given infusion (i.e. vehicle or drug) and the 191 preceding control period. Repeated-measures ANOVA (Holm-Sidak method) was used to test for 192 the significance of differences between vehicle/SKA-31 infusions and SNP. A P value <0.05 was 193 considered statistically significant. Except where noted, data are presented as mean ± SEM. 194 195 3. Results 196 Seven anesthetized, juvenile pigs were acutely implanted with blood pressure transducers 197 and Doppler flow probes that allowed us to measure mean aortic and inferior vena cava 198 pressures, systemic conductance and regional conductance in carotid, renal and coronary arteries. 199 Myocardial performance was monitored via a single lead electrocardiogram and implanted 200 sonomicrometry crystals in the myocardium to assess LV dimensions. Table 1 presents average 201 hemodynamic parameters in all 7 animals measured at baseline, following instrumentation and 202 recovery and before the first experimental infusion (saline). 203 204 3.1 SKA-31 Dose Response 205 Following surgical interventions, animals were allowed to recover until steady-state basal 206 levels of mean aortic pressure and heart rate were achieved. After a minimum 10 min period of 207 steady-state baseline recording, we commenced with the first (saline) infusion. Figure 1 displays 208 representative tracings of the effect of individual bolus administrations of saline, drug vehicle,

209 SKA-31 (0.1 – 5.0 mg/ml/kg) on mean aortic pressure (mPAO, panel A), systemic conductance 210 (panel B), measured conductance in carotid, coronary and renal arteries (panel C) and heart rate 8

211 (panel D). While SKA-31 infusions had clear effects on these hemodynamic parameters, neither 212 saline nor vehicle infusions had any observable effects. In an effort to benchmark the effects of 213 SKA-31 on the measured parameters, we infused a single dose of the well characterized 214 nitrovasodilator sodium nitroprusside (SNP) following recovery from the SKA-31 evoked 215 hemodynamic changes. As displayed on the right hand side of Figures 1A-D, SNP infusion (5.0

216 g/ml/kg) produced qualitatively similar changes in mPAO, systemic conductance, carotid, 217 coronary and renal artery conductances and heart rate when compared with SKA-31. In case of

218 mPAO (Fig. 1A), intravenous infusion of SKA-31 significantly decreased mPAO in a dose- 219 dependent manner versus each preceding control period, with the greatest decrease occurring

220 after the highest dose (5.0 mg/ml/kg) (Figure 2). SNP infusion also significantly decreased mPAO 221 and this change was comparable to that measured following infusion of 5.0 mg/ml/kg SKA-31. 222 As quantified in Figure 3, the time to peak response for the SKA-31 induced decrease in

223 mPAO was slowest at 0.1 mg/ml/kg drug administration and became faster with increasing 224 dosages. At a dosage of 5.0 mg/ml/kg, SKA-31 infusion resulted in a significantly faster decline

225 in mPAO compared with SNP.

226 In contrast to the observed decreases in mPAO, SKA-31 did not significantly alter mean

227 inferior vena cava pressure (mPIVC), compared with preceding control values (Figure 4). In the

228 case of SNP, we did observe a trend toward lower mPIVC, although this change did not reach 229 statistical significance. 230 231 3.2 Conductance and Resistance 232 Figure 5 shows the effect of SKA-31 and SNP administration on absolute changes in 233 systemic vascular resistance (SVR) (Fig. 5A), along with the calculated percent changes in 234 systemic conductance (Fig. 5B). We observed no changes in SVR versus the preceding control 235 values following infusions of saline, drug vehicle or lower dosages of SKA-31 (0.1 and 0.3 236 mg/ml/kg), whereas dosages of 1.0, 3.0 and 5.0 mg/ml/kg each significantly decreased systemic 237 resistance. SKA-31 at the highest dosage decreased SVR to a level comparable to that evoked by 238 5.0 g/ml/kg SNP. Predictably, the inverse relationships were observed for drug-induced 239 changes in systemic conductance (Fig. 5B). 240 In addition to its impact on systemic conductance, we also examined the effect of SKA- 241 31 on blood flow in select vascular regions. As shown in Figures 1C and 6, SKA-31 and SNP 9

242 infusions produced qualitatively similar effects on conductance in the right carotid artery

243 (Gcarotid), left anterior descending coronary artery (Gcoronary), and left renal artery (Grenal). SKA-31

244 increased Gcarotid at dosages of 3.0 and 5.0 mg/ml/kg and produced a maximal change in 245 conductance similar to that observed with 5.0 g/ml/kg SNP. In the left anterior descending

246 coronary artery, SKA-31 also significantly increased Gcoronary at doses of 3.0 and 5.0 mg/ml; the 247 increase evoked by the latter dose approximated that observed with SNP. Interestingly, neither 248 SKA-31 nor SNP significantly increased blood flow in the renal artery (compared with the 249 preceding control conductance values) and vasodilatory responses in this artery were generally 250 blunted compared with carotid and coronary vessels (Fig. 1C). 251 252 3.3 Cardiac Function 253 As depicted in Figure 7A, infusions of SKA-31 and SNP did not produce significant 254 changes in either cardiac stroke volume (SV) or heart rate (HR) under our experimental 255 conditions. We further examined potential drug-induced changes in the left ventricular end

256 diastolic area (ALVED), as measured by sonomicrometry crystals implanted in the septal wall and

257 LV endocardium, and the calculated LV stroke work (SWLV); both of these parameters are 258 expressed as the percent change from the respective preceding control value. Over the dosage

259 range of 1.0 to 5.0 mg/ml/kg, SKA-31 produced very modest decreases in ALVED (< 5% below

260 control), whereas SNP reduced ALVED by an average of 8% compared with control. In contrast to

261 the slight decreases observed in ALVED, SKA-31 evoked a clear, dose-dependent reduction in

262 SWLV over the range of 0.1 to 5.0 mg/ml/kg and produced a similar maximal decrease at a 263 dosage of 5.0 mg/ml/kg (-29%) as that observed following SNP infusion (-32%).

264 To evaluate in greater depth the observed decreases in SWLV following SKA-31 and SNP

265 administrations, we plotted the calculated percent changes in SWLV versus the observed percent

266 changes in mean aortic pressure (mPAO). The scatter plot in Figure 8 shows the relation between

267 SWLV and mPAO, based on the pooled data derived from all infusions of saline, vehicle, SKA-31 268 and SNP for the 7 animals employed in our study. Importantly, the calculated r2 value of 0.82

269 for the linear regression line indicates that more than 80% of the variance in SWLV can be

270 explained by the variance in mPAO. Using a similar approach, we also plotted the percent

271 changes in ALVED versus mPAO for all animals and infusions (i.e. saline, vehicle, drug) examined. 272 Linear regression analysis of this relation yielded a r2 value of only 0.24, indicating that no more 10

273 than 25% of the variance in ALVED can be explained by the variance in mPAO (P<0.001; data not 274 shown). Based on these results and the fact that drug infusions did not change stroke volume 275 under any condition (see Figure 7A), we conclude that neither SKA-31 nor SNP directly 276 impaired cardiac contractility. 277 278 3.4 Plasma Concentrations of SKA-31 Following Acute Infusion 279 In a separate group of 3 anesthetized and instrumented animals, we analyzed the plasma 280 concentrations of SKA-31 at select time points following acute intravenous infusion of a 3.0 281 mg/ml/kg SKA-31 bolus dose. Blood samples were withdrawn from the left jugular vein at ~1 282 min, 35 min and 75 min following complete infusion of the drug. The average free plasma 283 concentration of SKA-31 measured at each of the above time points (n = 3) was 77.5 ± 27.7 M,

284 27.3 ± 3.8 M and 27.4 ± 4.9 M, respectively. The volume of distribution for SKA-31 285 calculated from a semi-logarithmic plot of average SKA-31 plasma concentrations versus 286 sampling times was 0.19 L/kg. 287 288 4. Discussion 289 Using an anesthetized and instrumented porcine model, we have provided the first 290 detailed description of the systemic hemodynamic actions of SKA-31, a small molecule activator 291 of KCa 2.x and 3.1 channels [2], on key cardiovascular parameters in a large animal and how 292 these actions compare with those of SNP, an established nitrovasodilator and blood pressure- 293 lowering agent. As shown in Figures 1 and 2, intravenous administration of SKA-31 dose- 294 dependently evoked significant decreases in mean aortic pressure, with the highest dose utilized

295 in our study (5.0 mg/ml/kg SKA-31) producing a similar decrease in mPAO as that observed with 296 SNP (-23±3 and -28±4 mmHg, respectively) (Fig. 2B). In previous studies [2,5,8], acute in vivo 297 administration of SKA-31 was shown to lower blood pressure in both normotensive and 298 hypertensive mice and, more recently, Köhler and colleagues [4] have reported that acute 299 infusion of SKA-31 (0.4 and 2.0 mg/kg) transiently decreases systemic blood pressure in

300 conscious dogs. We also noted that the decrease in mPAO evoked by 5.0 mg/ml/kg SKA-31 was 301 more rapid compared with SNP (Fig. 3), even though both agents lowered mean aortic pressure

302 to a similar extent (Fig. 2). The slower time course of the SNP-mediated drop in mPAO may 303 reflect the fact that SNP requires vascular conversion/decomposition to release nitric oxide and 11

304 induce subsequent cellular actions in arterial smooth muscle [17], while SKA-31 directly 305 hyperpolarizes the endothelium by activating KCa channels. Collectively, these observations are 306 in agreement with the reported vasodilatory actions of SKA-31 in the intact coronary [7] and 307 skeletal muscle circulations [5,8] of rodents and the systemic circulation of the dog [4].

308 SKA-31 had no significant effect on mean inferior vena cava pressure (mPIVC) (Fig. 4). In

309 the case of SNP, we did observe a trend towards lower mPIVC, which would be in agreement with 310 the known clinical effects of SNP to lower , due to its ability to increase

311 venous capacitance [18]. One possible reason for our observation is that mPIVC was already quite 312 low under basal experimental conditions (~8 mmHg) and a further drug-induced decrease in

313 mPIVC may have been difficult to detect in our anesthetized pigs. Although KCa2.3 and KCa3.1 314 channel mRNA and whole cell K+ currents have been reported in venous endothelial cells (e.g. 315 HUVECs) [1,3,19], we are unaware of data describing a direct vasodilatory effect of KCa 316 channel activators on veins or the venous circulation. 317 SKA-31 dosages of 1.0 to 5.0 mg/ml/kg increased systemic arterial conductance, with the 318 highest dose producing an increase in conductance similar to that induced by SNP (Figure 5B). 319 We also observed increases in both carotid and coronary arterial conductances at 3.0 and 5.0 320 mg/ml/kg SKA-31 (Figure 6), which were similar to those observed with SNP at the highest 321 dosage of SKA-31. Interestingly, renal conductance appeared to be unaffected by either SKA-31 322 or SNP. In the case of SNP, this is somewhat unexpected, as other investigators have reported 323 that renal arteries are sensitive to [20,21]. The renal microcirculation is known 324 to exhibit strong autoregulatory behavior [22,23], which is critical for ensuring adequate blood 325 flow to glomerular units and protecting them from arterial pressure-induced damage. One 326 possible explanation for this apparent insensitivity of the renal conductance to SKA-31 and SNP 327 is that the renal circulation may have already been near-maximally dilated, due to a combination

328 of intrinsic and the somewhat lower mPAO present in our anesthetized pigs. 329 Alternatively, it is possible that reduced arterial resistance triggered an increase in peripheral 330 sympathetic tone to counteract reduced blood pressure, which then limited renal arterial dilation. 331 However, this possibility is less likely, as we observed no concomitant increase in heart rate with

332 declines in mPAO, which one would anticipate with the activation of a baroreceptor feedback 333 mechanism acting on the heart. 334 12

335 4.1 Cardiac Function 336 As small-conductance Ca2+-activated K+ channels have been reported in the atria and 337 pacemaker/conducting cells of murine and human cardiac tissue [24-27] and thus may be 338 activated in response to systemic SKA-31 administration, we recorded various indices of 339 myocardial performance during SKA-31 infusions. Importantly, we observed no significant 340 change in left ventricular stroke volume following administration of either SKA-31 or SNP. In 341 contrast to Köhler and coworkers [4], who reported a pronounced increase in heart rate (HR) 342 following acute SKA-31 infusion, we did not detect a significant change in HR in response to 343 SKA-31 or SNP infusions in the anesthetized pig (Fig. 7). The difference in HR responses in 344 these two studies could be attributed to the difference in experimental models, as Köhler and 345 colleagues examined conscious dogs (presumably with unsuppressed baroreceptor reflexes 346 providing autonomic nerve input to the heart) versus our anesthetized, instrumented pig model. 347 Importantly, the absence of SKA-31 induced changes in HR observed in our study strongly 348 suggests that plasma levels of SKA-31 sufficient to evoke substantial decreases in blood pressure 349 do not directly impact either pacemaker function or action potential propagation in the heart, as 350 revealed under conditions of minimal baroreceptor reflex activity. 351 Neither SKA-31 nor SNP significantly reduced central venous pressure (Fig. 4). One 352 possible explanation is that the relative magnitude of the arterial and venous effects of these 353 vasodilatory agents may differ [18,28-30] or an increase in total venous capacitance may have 354 been limited by a modest elevation in arterial capacitance as a result of evoked vasodilation. 355 Hemodynamically, a minor, undetected rise in total venous capacitance could explain the slight

356 decrease we observed in ALVED, our measure of left ventricular end-diastolic volume, in response 357 to SKA-31 and more so to SNP (Fig. 7B). Since left ventricular stroke work (SW) is a function 358 of both ventricular volume and pressure, the decreases observed in SW following administration

359 of SKA-31 and SNP could be explained, in part, by the minor reduction in ALVED. However,

360 further analysis of these data clearly showed that changes in ALVED accounted for less than 25%

361 of the variance in SW, whereas changes in mPAO accounted for more than 80%. Thus, the

362 observed decreases in SW could be largely attributed to the reductions in mPAO associated with

363 drug administration (Figure 8). Furthermore, the observed reductions in mPAO, indicative of left

364 ventricular , would be expected to offset the slight decreases in ALVED observed with 13

365 SKA-31 and SNP administrations, and the balance of these two effects would tend to maintain 366 stroke volume near control levels in the presence of either SKA-31 or SNP (Fig. 7A). 367 Our attempt to explore the pharmacokinetic behavior of SKA-31 revealed that its plasma 368 levels measured 35-75 min following intravenous infusion of a 3.0 mg/ml/kg dose were higher

369 than the reported EC50 values of SKA-31 for KCa3.1 channels (~0.3 M) and KCa2.3 channels 370 (~2 M) [2], suggesting that sustained activation of these endothelial channels might be 371 anticipated. However, the absence of prolonged hypotension following administration of SKA- 372 31 in our anesthetized pigs suggests that the relationship between the free plasma concentration 373 of SKA-31 (plasma protein binding of SKA-31 in mice and dogs is reported to be 35-40%) [2,4] 374 and its vasodilatory actions may not be a direct one and may be complicated by the availability 375 of additional drug binding sites or a more complex whole body distribution pattern. 376 Another explanation for the relatively short-lived hemodynamic response following 377 SKA-31 infusion could be a “desensitization” of the pharmacological targets for SKA-31 378 actions. Both KCa3.1 and KCa2.3 channels are subject to regulation by intracellular second 379 messengers or protein kinases and phosphatases. For example, phosphorylation of channel- 380 associated calmodulin by casein kinase 2 reduces the affinity of KCa2 channels for the

381 membrane phospholipid PIP2 [31] and likely contributes to the inhibition of KCa2 channel 382 activity by Gq-associated G-protein coupled receptors. KCa3.1 activity is increased by 383 phosphorylation of His358 in the channel’s C-terminus through the histidine kinase nucleoside

384 diphosphate kinase B (NDPK-B) [32], while the PI3P phosphatase myotubalarin related protein 6 385 (MTMR6) and the histidine phosphatase phosphohistidine phosphatase-1 (PHPT-1) inhibit 386 KCa3.1 function in T-cells [33,34]. Additionally, KCa3.1 currents can be regulated by cAMP- 387 dependent protein kinase (PKA) via Ser phosphorylation sites in the C-terminus [35-37], which 388 may impair endothelium-dependent vasodilation [38]. 389 Finally, the ability of a KCa channel activator to lower blood pressure more effectively in 390 hypertensive versus normotensive mice [2,5] suggests that this class of compound may be 391 beneficial in the acute or chronic treatment of elevated blood pressure. Our results showing that 392 SKA-31 reduces blood pressure and increases systemic conductance in the pig suggest that 393 translational studies examining the potential blood pressure-lowering actions of a KCa channel 394 activator in a large animal model of or vascular disease are likely feasible. 395 14

396 4.2 Summary and Conclusions 397 The results of our study demonstrate that the KCa channel activator SKA-31 effectively 398 and reversibly increases systemic conductance in a dose-dependent manner and lowers mean 399 aortic pressure in a large animal model. The observed hemodynamic actions of SKA-31 closely 400 mimic those evoked by SNP. SKA-31 did not directly affect cardiac contractility, nor did it 401 appear to impact heart rate or excitability. The common and overlapping cardiovascular 402 responses to SKA-31 and SNP are consistent with the conclusion that SKA-31 acts primarily on 403 blood vessels to evoke its effects on the systemic vasculature. Given that SKA-31 is strictly an 404 endothelium-dependent vasodilator [6], endothelial KCa channel activators may be useful as an 405 alternative pharmacologic strategy to evoke acute arterial vasodilation in situations where the 406 hemodynamic actions of SNP may not be desirable or effective (e.g. nitrate tolerance). 407 408 Acknowledgements 409 The authors would like to acknowledge the excellent surgical expertise of Ms. Cheryl Meek 410 throughout this study. This work was supported by research funding to A.P. Braun (Canadian 411 Institutes of Health Research MOP 97901), to H. Wulff (National Institutes of Health 412 NS072585) and to J.V. Tyberg ( Foundation of Canada/Pfizer Canada). 413 414 Conflict of Interest: On behalf of the all authors, the corresponding author states that no 415 conflicts of interest exist. 416 417 References 418 419 [1] Sheng J-Z, Ella S, Davis MJ, Hill MA, Braun AP. Openers of SKCa and IKCa channels 420 enhance agonist-evoked endothelial nitric oxide synthesis and arteriolar dilation. FASEB 421 J 2009;23: 1138-1145. doi: 10.1096/fj.08-120451 422 [2] Sankaranarayanan A, Raman G, Busch C, Schultz T, Zimin PI, Hoyer J et al. 423 Naphthol[1,2-d]thiazol-2-ylamine (SKA-31), a new activator of KCa2 and KCa3.1 424 potassium channels, potentiates the endothelium-derived hyperpolarizing factor response 425 and lowers blood pressure. Mol Pharmacol 2009;75: 281-295. doi: 426 10.1124/mol.108.051425 15

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538 539 Table 1 – Baseline hemodynamic parameters in anesthetized pigs immediately prior to the 540 control saline infusion at the start of the experiment. 541 HR (bpm) 122±9 SV (ml) 23±3

PLVED (mmHg) 11±1

mPAO (mmHg) 71±6

mPIVC (mmHg) 7±1

542

543 HR, heart rate in beats per minute; SV, left ventricular stroke volume; PLVED, end-diastolic left

544 ventricular pressure; mPAO, mean aortic pressure; mPIVC, mean inferior vena cava pressure. Data 545 represent the means ± SEM calculated from 7 pigs in total. 546 547 548 Figure Legends 549 Figure 1 – Representative data from one pig demonstrating the rapid and reversible effects of 550 SKA-31 and sodium nitroprusside (SNP) following acute intravenous infusion on mean aortic

551 pressure (mPAO) (panel A), systemic vascular conductance (panel B), measured conductance in 552 coronary, carotid and renal arteries (panel C) and heart rate (panel D). In each panel, the sections 553 of continuous data points displayed represent 5-min epochs that were extracted from the master 554 data record and illustrate the basal levels and evoked changes in the measured parameters in 555 response to the infusions. The horizontal bars and labels provided beneath each panel specify the 556 experimental infusion for the 5-min section of data appearing immediately above each 557 description. Note that all displayed data were acquired simultaneously during the experiment. 558 Individual infusions were separated by a 15-20 min recovery period (indicated by the breaks 559 between the sections of data points) and control hemodynamic data were acquired for the first 1- 560 2 min period immediately prior to a given infusion, once a steady baseline was clearly apparent 561 (not shown). 562 20

563 Figure 2 - Quantification of mean aortic pressure (mPAO) under control conditions and following 564 acute infusion of SKA-31 (0.1 – 5.0 mg/ml/kg) and SNP (5.0 µg/ml/kg) (panel A). Panel B

565 quantifies the drug-evoked changes in mPAO relative to the preceding control value for each 566 experimental condition. N = 7 animals for both panels A and B. 567

568 Figure 3 – Quantification of the time to maximal change in mean aortic pressure (mPAO) 569 following intravenous infusion of either SKA-31 (0.1 - 5.0 mg/ml/kg) or SNP (5.0 g/ml/kg).

570 Administration of either saline or drug vehicle did not evoke measurable changes in mPAO. The 571 response evoked by 5.0 mg/ml/kg SKA-31 was significantly faster than that elicited by SNP, as 572 determined by two-way ANOVA; P < 0.05, n = 7 animals. 573 574 Figure 4 – Lack of effect of SKA-31 (0.1 - 5.0 mg/ml/kg) on mean inferior vena cava pressure

575 (mPIVC) following acute administration. Histogram displays mPIVC values recorded in response 576 to infusions of saline, drug vehicle and the indicated dosages of SKA-31 and SNP. Values for

577 baseline mPIVC (control) immediately preceding each infusion are designated by the black bars. 578 579 Figure 5 – Acute administration of SKA-31 and sodium nitroprusside (SNP) reduce systemic 580 vascular resistance (SVR). Panel A displays absolute values for SVR recorded prior to a given 581 drug infusion and following SKA-31 and SNP infusions at the indicated dosages. For the latter 582 data, measurements were taken during the peak change in SVR. Panel B displays the calculated 583 percentage change in systemic vascular resistance under each infusion condition compared with 584 the preceding control. 585 586 Figure 6 – Quantification of evoked changes in arterial conductance calculated for the carotid, 587 coronary and renal arteries in response to infusions of saline, drug vehicle, SKA-31 (0.1 – 5.0 588 mg/ml/kg) and SNP (5.0 g/ml/kg). Histogram displays the percentage change in conductance in 589 each artery evoked by administered drugs relative to the preceding control value for each 590 infusion. Asterisks indicate a statistically significant difference compared with the baseline 591 conductance value preceding a given infusion. 592 21

593 Figure 7 – Quantification of the effects of acute administration of saline, drug vehicle, SKA-31 594 (0.1 – 5.0 mg/ml/kg) or SNP (5.0 µg/ml/kg) on left ventricular stroke volume and heart rate 595 (panel A). No significant changes were noted for either stroke volume or heart rate in response to 596 a given infusion compared with the values measured during the preceding control period. The

597 histogram in panel B displays the percentage changes in left ventricular area (ALVED) and stroke 598 work (SW), relative to the baseline values measured during the control period preceding each 599 indicated infusion. 600 601 Figure 8 – Scatter plot displaying the relation between observed changes in left ventricular stroke

602 work (SW) and mean aortic pressure (mPAO) following infusions of saline, vehicle, SKA-31 and

603 SNP. Percent changes in mPAO, along with accompanying percent changes in SW, were first 604 calculated in response to each infusion utilized in a given experiment. Data points from all 7 605 animals were then plotted against each other in a pair-wise fashion, as depicted by the individual 606 symbols on the graph. The straight line through the symbols represents a linear regression fit to 607 the pooled data points (r2 value = 0.82; P < 0.001). 608 Table 1 Click here to download high resolution image Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image Figure 5 Click here to download high resolution image Figure 6 Click here to download high resolution image Figure 7 Click here to download high resolution image Figure 8 Click here to download high resolution image